A universal screening assay for glycosynthases: Directed evolution of glycosynthase XynB2(E335G) suggests a general path to enhance activity

Glycosynthases are catalytic mutants of mainly retaining glycoside hydrolases that catalyze the synthesis of oligosaccharides from their corresponding glycosyl-fluoride, donors and suitable acceptors. Here we describe the development of a general, high-throughput screening procedure for glycosynthase activity, which is based on the release of hydrofluoric acid, a by-product of all glycosynthase reactions. This assay is sensitive, does not require the synthesis of special chromophoric or modified substrates, and, most importantly, is applicable for all glycosynthases. We used this screening procedure on error-prone PCR libraries to isolate improved glycosynthase variants of XynB2(E335G) glycosynthase, a family 52 beta-xylosidase from Geobacillus stearothermophilus. The improved variants exhibited higher K-M values toward the acceptor and the donor, suggesting that enzyme-product release is rate determining for k(cat).